A novel signal transduction pathway has recently been revealed in the interferon system. In this pathway, the regulatory subunits of a transcription factor, ISGF3, are directly phosphorylated by a tyrosine kinase(s) which is induced by interferon-alpha. Studies from this laboratory have shown that these regulatory subunits (p113 and p9l/84, ISGF3alpha) contain SH2 and SH3 domains, and that tyrosine phosphorylation is required for their activation. This is the first example of a transcription factor which can be activated directly by a tyrosine kinase. In this application, we propose investigating the detailed mechanism of activation of ISGF3. We will focus on the functional effects of tyrosine phosphorylation of ISGF3alpha proteins and the roles of the SH2 and SH3 domains in mediating interferon-alpha-induced signal transduction. We will further determine whether tyrosine kinase Tyk2 directly phosphorylates ISGF3alpha proteins. The nuclear localization signal (NLS), domains required for interactions between ISGF3 proteins and the stoichiometry of ISGF3 will also be characterized. We will determine whether this observed tyrosine phosphorylation unmasks the putative leucine zipper-like domains and NLS, resulting in complex formation and translocation of ISGF3alpha proteins to the nucleus.